1. Field of the Invention
This invention pertains to the field of high throughput assays for identifying modulators of interactions between the signal transducer and activator of transcription molecules STAT4 and STAT6 and their corresponding receptors.
2. Background
The growth, differentiation, and functional responses of many cells are controlled by cytokines, growth factors, and hormones. While many of these responses are desirable for normal development of an organism, or to help an organism fight an attacking pathogenic organism or a disease condition, in other situations the cellular response to a given stimulus is inappropriate. For example, T helper cells are an integral part of an organism's defense against harmful antigens. However, when produced at an inappropriate time, T helper cells can attack the organism itself, thus resulting in an autoimmune disease. In other situations, it may be desirable to trigger or enhance a particular response, even in the absence of the usual stimulatory signal.
Development of methods by which one can influence the response of a cell to a given cytokine or growth factor stimulus would be facilitated by an understanding of the signal transduction pathway by which an interaction between a cell and a cytokine is translated into a response by the cell. Generally, cytokines bind to a cell surface receptor of the cytokine receptor superfamily. Binding of a cytokine to its corresponding receptor causes receptor aggregation as well as association of a Janus protein tyrosine kinase (Jaks) with the receptor. Receptor aggregation allows the Jaks to transphosphorylate each other, which dramatically increases their catalytic activity. The activated Jaks then phosphorylate the receptor. Also phosphorylated by the activated Jaks is a polypeptide that is responsible for transmitting the signal to the cell's nucleus. These polypeptides are members of a family of proteins known as Signal transducers and activators of transcription (STAT; Darnell et al. (1994) Science 264: 1415; for review, see, e.g., Ihle et al. (1994) Trends Biochem. Sci. 19: 222; Ihle et al. (1995) Trends Genetics 11: 69). STATs are activated by contact with the phosphorylated receptor; activation results in the STAT polypeptides forming a dimer and entering the nucleus, where the STAT dimer binds to the regulatory region of a gene that is inducible by the particular cytokine. Binding of the activated STAT dimer triggers transcription of the gene.
Seven STAT polypeptides are known (STAT1, STAT2, STAT4, STAT5a, STAT5b, and STAT6); these polypeptides have molecular masses from 84-113 kDa. Each STAT protein contains a Src homology-2 (SH2) domain capable of recognizing one or more phosphotyrosine sequences in the cytoplasmic portion of the activated receptor (Shuai et al. (1993) Nature 366: 580). Each cytokine receptor is specific for a particular STAT protein, and each STAT activates transcription of certain genes, thus providing two layers of cytokine specificity.
At least two of the STAT polypeptides, STAT4 and STAT6, are intimately involved in regulation of immune responses. STAT4 transduces to the nucleus signals from the IL-12 receptor. IL-12 is involved in the development of a T.sub.H 1 immune response (Kaplan et al. (1996) Nature 382: 174-177), which is part of an organism's defense against intracellular pathogens. IL-12 is also necessary for the T-cell-independent induction of the cytokine interferon (IFN)-.gamma., which is a key step in the initial suppression of bacterial and parasitic infections. Knockout mice which lack STAT4 were found to be defective in all IL-12 functions tested, including the induction of IFN-gamma, mitogenesis, enhancement of natural killer cytolytic function and T.sub.H 1 differentiation (Thierfelder et al. (1996) Nature 382: 171-174).
IL-4 signals are transduced to the nucleus by STAT6. IL-4 is a key cytokine in the initiation of a T.sub.H 2 immune response, and also activates B and T lymphocytes. STAT6-deficient mice were deficient in IL-4 activities (Kaplan et al. (1996) Immunity 4: 313-319; Takeda et al. (1996) Nature 380: 627-630; Shimoda et al. (1996) Nature 380: 630-633).
Because of the importance of STAT4 and STAT6 in modulating the immune response of an organism, both in response to infection and in undesirable conditions such as inflammation, allergic reactions, and autoimmune diseases, a need exists by which the clinician can enhance or reduce STAT4 and STAT6 signals. Intervention at the STAT level would have significant advantages compared to previous approaches, which typically target the IL-4 or IL-12 cytokine itself, or the interaction of the cytokine with the receptor. Disruption of cytokine function itself can cause a variety of undesirable side effects. These can be avoided by intervening at the level of STAT-mediated signal transduction. However, identification of agents that can modulate STAT4 and STAT6-mediated signal transduction has been hampered by the lack of suitable assays. Assay of binding of STAT4 and STAT6 to their corresponding receptors, and identification of agents which increase or decrease the degree of such binding, has been difficult because of the high rate at which the STAT4 and STAT6 polypeptides bind to (k.sub.on) and leave the receptor (k.sub.off) after initial binding. The present invention fulfils this need for effective assays by which modulators of STAT4 and STAT6 binding can be identified, as well as other needs.